The deamination mechanism of the 5,6-dihydro-6-hydro-6-hydroxylcytosine and 5,6-dihydro-5-methyl-6-hydroxylcytosine under typical bisulfite conditions

The hydrolytic deamination of HOHCytN3⁺ (paths A and B), and 5-HOHMeCytN3⁺ (paths C and D) have been explored in the presence of the ${\mathrm{HSO}}_3^{-}$ group. The activation free energy barriers (ΔG^s≠) of paths A and C show significant difference, which increases the possibility in differentiating cytosine (Cyt) from 5-methylcytosine (5-MeCyt) under bisulfite conditions. Meanwhile, the calculated rate constants with Wigner tunnelling correction of the rate-limiting step is much lower for path A than for path C, and by extrapolation, the difference between Cyt and 5-MeCyt for both modified pyrimidine base may have a greater chance by bisulfite treatment.